Method for detecting chemical substances in samples of material that can be taken from a subject, in particular for detecting embryotoxic factors

ABSTRACT

A method for detecting chemical substances in samples of material that can be taken from a subject, comprising a step of preparing a sample of a substance coming from a donor organism; a step of associating with said sample of a substance a control element suitable for verifying a presence of at least one active agent in the sample of the substance and a step of verifying the presence or absence of the active agent based on a change in state in said control element; the control element comprising at least one living organism having the ability to nourish itself autonomously through temporary organs associated with the living organism itself and able to supply nutritive substances to the latter without taking said nutritive substances from an outside environment.

The present invention relates to a method for detecting, in samples of material that can be taken from a subject (who can be, for example, a woman in fertile age), one or more chemical substances which can exert different effects, such as a harmful effect on the growth or maintenance of the viability of an embryo.

As is known from the clinical statistics available today, approximately 40% of patients affected by phenomena of recurrent miscarriage with no ascertained cause are characterized by the presence of so-called “embryotoxic factors” (abbreviated with the acronym ETFs) in their blood: these factors are a set of molecules correlated with a biological mechanism whereby during the implantation stage or during the earliest stages of pregnancy, the embryo is mistakenly considered a foreign object in the future mother's body, which develops a specific immune response with the aim of eliminating it.

Also known from recent clinical studies is the correlation between the presence of ETFs in the mother's body and a non-negligible number of cases of idiopathic infertility (or even cases of sterility mistakenly attributed to so-called endometriosis), and this is indicative of the central role of ETFs as “markers” of various clinical risk situations.

In other words, it is of fundamental importance to implement systems for diagnosing ETFs, so as to be able to predict difficulties in embryo implantation and/or the occurrence of miscarriages or anything else in advance: in this regard, the prior art available today (still considered at an experimental stage) envisages the use of mouse embryos or live cell cultures.

Irrespective of the type of test media utilizable, the known methods use a blood sample of a patient, from which the white cells are extracted and then cultured in the presence of embryonic factors that ensure their activation (or, in other words, stimulate an immune response), and subsequently the so-called “conditioned medium” (abbreviated in laboratory jargon as CM) obtained from the extraction/activation of white blood cells is placed in contact with the organic substances used for analysis (for example, if mouse embryos are used, the latter are observed for three days after being incubated with the CM).

Following this exposure, if the embryos die, it is deduced that toxic substances are present in the patient's serum (these in general fall within the definition of ETFs), whereas if, on the contrary, the embryos develop normally, the test is negative.

Alternatively, the CM is incubated with a JEG-3 type cell culture and the level of cell mortality is evaluated after three days (this level is considered indicative of the presence of ETFs).

The methods for verifying the presence of ETFs summarized above have several substantial disadvantages, particularly in terms of operating costs, speed of execution of the protocol and hence of production of results, low statistical “robustness” and operational flexibility: in particular, the choice of a “biological material ” (mouse embryos or JEG-3 cells) to react the culture medium with implies operational complications, both from a cost standpoint and in terms of the growth/culture/development of the test organic material itself.

Moreover, the use of “animal models” in the known test processes could lead to a problem of an ethical type, given the growing attention/aversion of the media and public opinion precisely toward the use of animal models in clinical trial processes: in the case of tests to identify ETFs (tests that have evident diagnostic purposes), a possible limitation/abolition “by law” of animal models in this type of testing as well would lead, in fact, to a nearly complete impossibility of carrying them out, also given that such tests for identifying ETFs cannot be considered like tests performed under clinical trial conditions (where the use of animal models may and may continue to be allowed), as they are veritable diagnostic tests.

The object of the present invention is thus to conceive a method for detecting substances, and in particular toxic substances, such as, for example, ones tied to the definition of ETFs, which overcomes the disadvantages of the prior art.

In particular, it is an object of the present invention to implement a method that minimizes operating costs, provides a large amount of material to be exposed to the possible presence of ETFs and thus makes it possible to carry out a large number of tests (possibly also of a recursive or parallel type on samples taken from the same subject) with excellent qualitative and quantitative reliability in terms of results.

These and other objects of the invention are achieved with a method illustrated here below, in a non-limiting example embodiment thereof, as well as in one or more of the appended claims.

The method substantially comprises the typical steps of a laboratory test procedure, namely:

preparing a sample of a substance (which can typically comprise organic fluids and/or portions of tissues originating from the body of a donor);

associating with the sample of a substance a control element suitable for verifying a presence of at least one active agent in the sample of the substance; and

verifying a presence or absence of the active agent based on a change in state (physical, chemical or biological) in the control element,

Advantageously, the present method envisages that the control element comprises at least one living organism having the ability to nourish itself autonomously through temporary organs associated with the living organism itself and able to supply nutritive substances to the latter, that is, without taking them from an outside environment.

The particular definition of this property of the control element in accordance with the present invention makes it possible to use an organism with a sufficient degree of tissue and systemic development, such that from a regulatory standpoint the material is not considered “animal”, but only a “biological material”, and thus does not fall within the area (that of experimentation) which is regulated by legislation on tests with animals: this will be better explained further below in the present description.

Going into detail, one can see how the living organism selectable in accordance with the present method can comprise at least a fish belonging to the class of Actinopterygii, and typically the species called Danio rerio (also known by the common name “zebrafish”): in even further detail, one can choose a Danio rerio belonging to the “AB” type genotypic strain.

Alternatively, the present method can be implemented by choosing a fish of the species Oryzias latipes (also known by the common name “Japanese rice fish” or by the name “medaka”) as the living organism for performing tests and analyses.

Or, as an alternative to the models described above, the class of amphibians including the genus Xenopus.

According to a particular feature of the present invention, the possible organisms (such as, for example, the two fish and the amphibian presented above) will be used in their biological form in which they are able to nourish themselves autonomously by means of a temporary organ referred to in jargon as “yolk sac” (otherwise called simply yolk according to current English scientific terminology) or similar temporary organs that have developed integrally with the living organism itself: if fish of the species Danio rerio are used, this is possible until the living organism reaches a lifetime less than or equal to 120 hours of development after fertilization (at a standard temperature), whereas in the case in which fish of the species Oryzias latipes are used, this is possible until the living organism reaches a lifetime less than or equal to 288 hours of development after fertilization. In the case of Xenopus, the larval stage is aquatic and the yolk is preserved for up to about 96 hours of development after fertilization. After this stage it draws nourishment from the outside.

Xenopus is considered by the EU to be an official experimental animal model.

At this point it should be noted that upon exceeding this time threshold, the embryo of Danio rerio (or Oryzias latipes or Xenopus, depending on the cases) uses up all the nutrients present in its yolk sac and becomes dependent on the outside environment from a nutritional viewpoint: from this moment on it is considered an animal from a regulatory viewpoint and is consequently subject to legislation on animal experimentation (which entails considerable increases in operating costs and in costs of executing laboratory tests on a large scale).

From a practical viewpoint, the step of associating a control element with the sample of a substance comprises the following sub-steps:

first, a predetermined number of living organisms is exposed to the sample of the substance taken (for example, one may consider at least 20 embryos of Danio rerio having a development time of no more than 72 hours after fertilization);

equal groups of embryos (of Danio rerio or Oryzias latipes or Xenopus, for example two groups of 10) are placed in a specific culture medium in suitable verification containers, which can be, for example, two adjacent wells of a culture plate (which can in turn comprise 24 wells); and

the sample of the substance, which can typically be a blood serum taken from a donor subject, is added to the culture medium just mentioned at a suitable concentration.

As regards the execution of the step of verifying the presence/absence of the active agent, it should be noted that this can conveniently be designed to verify an absence/presence of so-called “embryotoxic factors (ETFs)” and comprise the following sub-steps:

an environmental interaction is maintained, typically in the wells of the culture plate, between the embryos and the sample of the substance for a period of interaction defined as twelve hours (or in any case multiples of twelve hours, up to a maximum of 48 hours);

a number of deceased embryos is counted after the just mentioned twelve hours and/or above-defined multiples of twelve hours have elapsed; and

a level of toxicity is attributed, which is proportional and quantitatively correlated to a level of chemical activity of the embryotoxic factors (ETFs) present in the sample of the substance.

In accordance with the present method, a classification of the sample of a substance can be defined according to the following parameterization:

the toxicity level of the sample of a substance is defined as “toxic” when the number of dead embryos exceeds 50% of the total number of embryos placed in the verification containers;

the toxicity level of the sample of a substance is defined as “moderately toxic” when the number of dead embryos ranges between 50% and 30% of the total number of embryos placed in the verification containers; or “non-toxic” when the number of dead embryos is less than or equal to 20% of the total number of embryos placed in the verification containers.

From the viewpoint of the operational possibilities available after the step of testing and qualitatively/quantitatively determining the toxicity level, it is possible to act upon the donor subject based on the result of the step of verifying the presence/absence of embryotoxic factors (ETFs):

conveniently, the possible actions upon the donor subject can consist in exposing the donor subject to immune system-suppressing agents, for example by means of:

infusions of immunoglobulins and/or of suppressors of cytokine production (typically intravenously);

topical applications of progesterone and/or progesterone oil on the donor subject or on a culture medium suitable for hosting processes of artificial fertilization associated with the donor subject; and/or

administration of vitamin or non-vitamin or nutraceutic compounds or natural and/or phytotherapeutic active ingredients as well as of other food supplements.

The invention achieves various advantages, above all in terms of optimizing the ratio between operating costs and quality of the results obtained by the tests.

In particular, the possibility of developing large quantities of “biological material”, considered such in accordance with the invention, makes it possible to have, in shorter times and with reduced, economical culture spaces, a “test field” made up of a very large number of specimens that can act as “biological material”, and consequently ensure faster execution times (as well a greater reliability of the toxicological tests).

Moreover, the use of so-called “biological material” according to the formal definition applied today in biological laboratory practices (rather than the use of embryos as legally classified under current legislation) allows one to avoid being subject to the complex and restrictive rules of animal experimentation, making the entire process faster and more efficient and increasing both the productivity of the laboratory and the possibility of carrying out a larger number of tests.

The method according to the present invention is thus carried out on zebrafish embryos that have developed for no more than 120 hours after fertilization (or, alternatively, medaka embryos that have developed for no more than 288 hours after fertilization or Xenopus embryos which cannot have developed for more than about 96 hours after fertilization), and which within this time limit are not classifiable as “animal” according to current legislation: at the same time, by using organisms belonging to the species Danio rerio (or similar species, such as the ones described above) it is conveniently possible to expose the live organs/systems undergoing formation to the possible presence of ETFs, thereby obtaining complete only feedback through an accurate and realistic response (which is typically obtained by an “in vivo” model). 

1. A method for detecting chemical substances in samples of material that can be taken from a subject, said subject being human or animal, in particular for detecting embryotoxic factors, comprising the following steps: preparing a sample of a substance, said sample of a substance preferably comprising organic fluids and/or portions of tissues coming from a donor organism; and associating with said sample of a substance a control element suitable for verifying a presence of at least one active agent in said sample of a substance; and verifying a presence or absence of said active agent based on a change in state in said control element, characterised in that said control element comprises at least one living organism having the ability to nourish itself autonomously through temporary organs associated with the living organism itself and able to supply nutritive substances to the latter without taking said nutritive substances from an outside environment.
 2. The method according to claim 1, wherein said living organism comprises at least one fish belonging to the class Actinoptetygii and the genus Xenopus of the class Amphibia.
 3. The method according to claim 2, wherein said fish is of the species Danio rerio or “zebrafish” and is preferably a Danio rerio belonging to a strain of genotype “AB”, said fish even more preferably having a lifetime less than or equal to 120 hours of development and/or cell multiplication after fertilization.
 4. The method according to claim 2, wherein said fish is of the species Oryzias Latipes or “medaka”, said fish preferably having a lifetime less than or equal to 288 hours of development and/or cell multiplication after fertilization.
 5. The method according to claim 1, wherein said living organism is able to feed itself autonomously via a temporary organ of the “yolk sac” type or the like, said temporary organ being developed integrally with the living organism.
 6. The method according to claim 1, wherein said step of associating a control element with said sample of a substance comprises the following sub-steps: exposing a predetermined number of living organisms to said sample, said predetermined number of living organisms preferably being at least 20 embryos of Danio rerio having a development and/or cell multiplication time no greater than 72 hours after fertilization; placing equal groups of said embryos of Danio rerio, preferably in a number equal to 10, in respective verification containers; adding the sample of a substance to said verification containers, said sample of a substance comprising blood serum and/or plasma taken from a donor subject.
 7. The method according to claim 6, wherein said verification containers comprise at least two adjacent wells of a culture plate, said culture plate preferably comprising a total of 24 wells.
 8. The method according to claim 1, wherein said step of verifying a presence or absence of said active agent is designed to verify an absence or presence of embryotoxic factors (ETFs) and comprises the follow sub-steps: maintaining an environmental interaction, preferably in said verification containers of said culture plate, between the embryos and the sample of a substance for a defined period of interaction ranging from twelve hours or, preferably, multiples of twelve hours, up to a maximum of 48 hours; counting a number of deceased embryos after said twelve hours and/or after said multiples of twelve hours have elapsed; and attributing to the sample of a substance a toxicity level that is proportional and quantitatively correlated to a quantitative presence or a level of chemical activity of said embryotoxic factors (ETFs).
 9. The method according to claim 8, wherein said toxicity level is defined as “toxic” when the number of dead embryos exceeds 50% of the total number of embryos placed in said verification containers of the culture plate, said toxicity level being alternatively defined as “moderately toxic” when the number of dead embryos ranges between 50% and 30% of the total number of embryos placed in said verification containers of the culture plate, and said toxicity level being alternatively defined as “non-toxic” when the number of dead embryos is less than or equal to 20% of the total number of embryos placed in said verification containers of the culture plate.
 10. The method according to claim 1, wherein there is also a step of operating on the donor subject based on a result of said step of verifying the presence or absence of embryotoxic factors (ETFs), said step of operating on the donor subject comprising exposure to immune system-suppressing agents and preferably comprising: infusions of immunoglobulin and/or suppressors of cytokine production, still more preferably administered intravenously; and/or topical applications of progesterone and/or progesterone oil on the donor subject or culture medium suitable as a host for artificial insemination processes associated with the donor subject; and/or administering vitamin or non-vitamin or nutraceutic compounds or natural and/or phytotherapeutic compounds as well as other food supplements.
 11. The method according to claim 2, wherein said amphibian is of the genus Xenopus, said amphibian preferably having a lifetime less than or equal to 96 hours of development and/or cell multiplication after fertilization. 